Secondary Assay for lipid storage modulators. NEFA Incorporation/Release.
Assay Submitter (PI): Carole Sztalryd; GRECC/Geriatrics, Veterans Affairs Medical Center, Department of Medicine, School of Medicine, University of Maryland, Baltimore, Maryland, USA. Beller, Mathias; Max-Planck-Institut fur Biophysikalische Chemie, Germany. ..more
BioActive Compound: 1
Data Source: University of Maryland
BioAssay Type: Secondary
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
Assay Submitter (PI): Carole Sztalryd; GRECC/Geriatrics, Veterans Affairs Medical Center, Department of Medicine, School of Medicine, University of Maryland, Baltimore, Maryland, USA. Beller, Mathias; Max-Planck-Institut fur Biophysikalische Chemie, Germany.
NCGC Assay Overview: Storing lipids as a reservoir for energy or the anabolism of elementary metabolites is a common feature of probably all cells and is conserved from bacteria to humans. The universal cellular lipid storage organelle is the so-called lipid storage droplet (LD). Although ubiquitous, LDs share a simple structure composed of a hydrophobic core that harbors the storage lipids, which is shielded by a droplet-specific phospholipid monolayer to which proteins are attached. The current model of LD biogenesis involves an incorporation of the lipid core into the membrane leaflets of the endoplasmic reticulum (ER) followed by a subsequent budding-like maturation of a LD, which ultimately pinches off. Once released, LD volume can increase by localized lipogenesis or fusion of existing droplet. Storage lipids are re-mobilized enzymatically by lipase activity. Lipase regulation in the adipocyte is heavily studied and involves multiple components including catecholamine signaling, the LD-associated proteins Perilipin and comparative gene identification (CGI-58) and at least two lipases named hormone sensitive lipase (HSL) and adipocyte triglyceride lipase (ATGL). LD regulation outside of the adipocyte is poorly understood and only few components are known. However, there is an urgent need to learn more about ectopic fat depots as mislocalized storage of lipids, for example in the liver or muscle, is an eminent health problem associated with insulin resistance or the metabolic syndrome. We developed a laser-scanning cytometer assay to enable 1,536-well screening and combined the results of the small molecules screen with an RNAi database based on lipid storage . To support evolutionary conservation of COPI effects on lipid storage we also utilized Exo1 in a mammalian cell system (AML12 cells). Exo1 reduced non-esterfied fatty acids (NEFA) release to the same extent as the siRNAs targeting COPI subunit encoding mRNAs.
Assay Protocol Summary:
Measurements of NEFA released from lipid droplets or incorporated into the TG fraction were performed as previously described . Briefly, AML12 cells treated with or without specific siRNAs (10 nM) for 4 d were incubated overnight with growth medium supplemented with 400 uM oleic acid complexed to 0.4% bovine serum albumin to promote triacylglycerol deposition and [3H] oleic acid, at 1 x 106 dpm/well, was included as a tracer. In lipolysis experiments, re-esterification of fatty acids in AML12 cells was prevented by including 10 uM Triacsin C (Biomol), an inhibitor of acyl coenzyme A synthetase, in the medium. Quadruplicate wells were tested for each condition. Lipolysis was determined by measuring radioactivity released into the media in 1 h. For the lipid extraction and thin layer chromatography, the cell monolayer was washed with ice-cold PBS and scraped into 1 ml of PBS. Lipids were extracted by the Bligh-Dyer method, and 10% of the total lipid
was analyzed by thin layer chromatography. AML12 cells treated with or without specific siRNAs were additionally incubated with either vehicle (DMSO), 5 uM of Exo1 (12.5 mg/ml DMSO), or BFA (10 mg/ml DMSO) during the time of radioactivity release into the
media (2 h). NEFA incorporation into the TG fraction and NEFA release are calculated as nM/mg protein. Protein measurements were performed using a commercial BCA assay kit (Pierce Biotechnology) according to the manufacturer's instructions. Statistical significance was tested by unpaired Student t test (GraphPad software).
NEFA Incorporation and NEFA Release Measured in AML12 Cells after compound treatment. Relative activity [(experimental/ALLStars negative control (control) and DMSO) Radiolabel assays for NEFA release (nM) relative to total protein concentration in cells treated with siRNAs targeting the indicated transcripts in the presence of DMSO only or Exo1 (5 uM) in DMSO Data was determined to be significance at p < 0.01, unpaired t-test. Standard error is indicated.
One compounds was found active and assigned a score of 100. Inactive compounds were assigned a score of 0.
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Data Table (Concise)